Gain-of-function experiments have demonstrated the potential of Notch signals to expand primitive hematopoietic progenitors, but whether Notch physiologically regulates hematopoietic stem cell (HSC) homeostasis in vivo is unclear. To answer this question, we evaluated the effect of global deficiencies of canonical Notch signaling in rigorous HSC assays. Hematopoietic progenitors expressing dominant-negative Mastermind-like1 (DNMAML), a potent inhibitor of Notch-mediated transcriptional activation, achieved stable long-term reconstitution of irradiated hosts and showed a normal frequency of progenitor fractions enriched for long-term HSCs. Similar results were observed with cells lacking CSL/RBPJ, a DNA-binding factor that is required for canonical Notch signaling. Notch-deprived progenitors provided normal long-term reconstitution after secondary competitive transplantation. Furthermore, Notch target genes were expressed at low levels in primitive hematopoietic progenitors. Taken together, these results rule out an essential physiological role for cell-autonomous canonical Notch signals in HSC maintenance.

Jagged1-mediated Notch signaling has been suggested to be critically involved in hematopoietic stem cell (HSC) selfrenewal. Unexpectedly, we report here that inducible Cre-loxP-mediated inactivation of the Jagged1 gene in bone marrow progenitors and/or bone marrow (BM) stromal cells does not impair HSC selfrenewal or differentiation in all blood lineages. Mice with simultaneous inactivation of Jagged1 and Notch1 in the BM compartment survived normally following a 5FU-based in vivo challenge. In addition, Notch1-deficient HSCs were able to reconstitute mice with inactivated Jagged1 in the BM stroma even under competitive conditions. In contrast to earlier reports, these data exclude an essential role for Jagged1-mediated Notch signaling during hematopoiesis. IntroductionHematopoietic stem cells (HSCs) exhibit self-renewing capacity as well as the ability to give rise to more committed progenitors that differentiate into all hematopoietic lineages. 1 The molecular mechanisms regulating stem cell self-renewal and/or differentiation are only poorly understood. Among the proteins that have been postulated to be involved in hematopoietic stem cell maintenance are the Notch receptors and their ligands. 2 Mammals have 4 Notch receptors (Notch1-4) that bind 5 different ligands (Jagged1-2, Delta-like 1-3-4). Expression of a constitutively active form of Notch1 (N1) in murine bone marrow progenitors can lead to increased HSC-self-renewal 3 or to the immortalization of stem cell like progenitors capable of undergoing lymphoid and myeloid differentiation both in vitro and in vivo. 4 In addition, coculture of murine or human HSCs with immobilized Notch ligands, or feeder cells expressing such ligands, can maintain or even enhance HSC self-renewal. [5][6][7][8][9] Recently, osteoblasts expressing the Notch ligand Jagged1 (J1) were identified as being part of the hematopoietic stem cell niche. Osteoblast-specific expression of the activated parathyroid hormonerelated protein receptor results in increased numbers of osteoblasts expressing high levels of J1. The increase in osteoblasts correlates with an increase in the number of HSCs, with evidence of N1 activation in vivo. 10 These results were interpreted to mean that J1-expressing osteoblasts regulate HSC homeostasis through N1 activation.To definitively assess the role of J1 in the hematopoietic system, we have generated inducible gene-targeted mice for J1. Surprisingly, inactivation of J1 in either bone marrow (BM) progenitors or BM stromal cells had no effect on HSC maintenance. In addition, N1-deficient HSCs transplanted into mice with inactivated J1 in the BM stroma reconstituted BM chimeras normally. Our data exclude an essential contribution of J1-mediated N1 signaling for HSC self-renewal or differentiation. Study design Generation and conditional inactivation of mice with a loxP-flanked J1A J1 genomic clone was isolated from a mouse genomic library using an oligonucleotide complementary to part of the first coding exon. LoxP sites were introduced into an XhoI site a...

Notch1 (N1) receptor signaling is essential and sufficient for T cell development, and recently developed in vitro culture systems point to members of the Delta family as being the physiological N1 ligands. We explored the ability of Delta1 (DL1) and DL4 to induce T cell lineage commitment and/or maturation in vitro and in vivo from bone marrow (BM) precursors conditionally gene targeted for N1 and/or N2. In vitro DL1 can trigger T cell lineage commitment via either N1 or N2. N1- or N2-mediated T cell lineage commitment can also occur in the spleen after short-term BM transplantation. However, N2–DL1–mediated signaling does not allow further T cell maturation beyond the CD25+ stage due to a lack of T cell receptor β expression. In contrast to DL1, DL4 induces and supports T cell commitment and maturation in vitro and in vivo exclusively via specific interaction with N1. Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak. Interestingly, preferential N1–DL4 binding reflects reduced dependence of this interaction on Lunatic fringe, a glycosyl transferase that generally enhances the avidity of Notch receptors for Delta ligands. Collectively, our results establish a hierarchy of Notch–Delta interactions in which N1–DL4 exhibits the greatest capacity to induce and support T cell development.